Carswell et al. found that the sera of mice infected with bacillus Calmette-Gubrin (BCG) and then treated with endotoxin contain a substance which necrotizes transplanted Meth A sarcoma; and named it tumor necrosis factor (hereinafter referred to as TNF) [Proc. Nat. Acad. Sci., U.S.A., 72, 3666 (1975)).
TNF is considered to be a physiologically active substance released from macrophages, and is known to be characterized in that
(i) when it is administered to animals bearing a certain kind of tumor (for example, Meth A sarcoma), it causes necrosis in the tumor and cures the animals; (ii) it has a cytotoxic effect in vitro on a certain kind of tumor cells (such as mouse L cells) but has scarcely any injurious effect on normal cells; and (iii) its activity is not animal species-specific.
Because of these characteristics, it has been strongly desired to develop TNF as a new type of antitumor agent.
TNF or a TNF-like substance has been reported in the following literature references or published in the following patent documents.
Green et al., Proc. Nat. Acad. Sci., U.S.A., 73, 381 (1976). PA1 Matthews et al., Br. J. Cancer, 42, 416 (1980). PA1 Ruff et al., J. Immunol., 125, 1671 (1980). PA1 Mannel et al., Infect. Immunity, 28, 204 (1980). PA1 Haranaka et al., Japan. J. Exp. Med., 51, 191 (1981). PA1 European Patent Publication No. 90892. PA1 European Patent Publication No. 86475. PA1 Japanese Patent Publication No. 21621/1983. PA1 A: adenine PA1 C: cytosine PA1 G: guanine PA1 T: thymine PA1 Ala: alanine PA1 Arg: arginine PA1 Asn: asparagine PA1 Asp: aspartic acid PA1 Cys: cysteine PA1 Gln: glutamine PA1 Glu: glutamic acid PA1 Gly: glycine PA1 His: histidine PA1 Ile: isoleucine PA1 Leu: leucine PA1 Lys: lysine PA1 Met: methionine PA1 Phe: phenylalanine PA1 Pro: proline PA1 Ser: serine PA1 Thr: threonine PA1 Trp: tryptophan PA1 Tyr: tyrosine PA1 Val: valine PA1 DNA: deoxyribonucleic acid PA1 cDNA: complementary DNA PA1 sscDNA: single-stranded cDNA PA1 dscDNA: double-stranded cDNA PA1 RNA: ribonucleic acid PA1 mRNA: messenger RNA PA1 poly(A)mRNA: poly(A)-containing mRNA PA1 dATP: deoxyadenosine triphosphate PA1 dCTP: deoxycytidine triphosphate PA1 dGTP: deoxyguanosine triphosphate PA1 dTTP: deoxythymidine triphosphate PA1 oligo(dC): oligodeoxycytidylic acid PA1 oligo(dG): oligodeoxyguanylic acid PA1 oligo(dT): oligodeoxythymidylic acid PA1 poly(A): polyadenylic acid PA1 poly(U): polyuridylic acid PA1 poly(dC): polydeoxycytidylic acid PA1 poly(dG): polydeoxyguanylic acid PA1 ATP: adenosine triphosphate PA1 EDTA: ethylenediaminetetraacetic acid PA1 kb: kilobases PA1 kbp: kilobase pairs PA1 bp: base pairs PA1 Meth A sarcoma: methylcholanthrene-induced sarcoma PA1 TNF: tumor necrosis factor PA1 rHu-TNF: recombinant human TNF PA1 A is a polypeptide of the formula [Ia] below in which one or more amino acids may be deleted or replaced by other amino acid(s), PA1 B is a peptide of the formula [Ib] below in which one to three amino acids may be deleted or replaced by other amino acid(s), PA1 X is a polypeptide, PA1 Y is Met, and PA1 m, n and p are 1 or 0; PA1 (9) a base sequence of the 577th to the 708th bases in the upper rows of Table 5, and especially PA1 (10) a base sequence of the 658th to the 708th bases in the upper rows of Table 5 PA1 A is a polypeptide of the formula [Ia] below in which one or more amino acids may be deleted or replaced by other amino acid(s), PA1 B is a peptide of the formula [Ib] below in which one to three amino acids may be deleted or replaced by other amino acid(s), PA1 X is a polypeptide, PA1 Y is Met, and PA1 m, n and p are 1 or 0; PA1 A. Inserting the DNA having or containing a base sequence encoding human TNF polypeptide or its principal portion, or its modified base sequence into an expression vector, PA1 B. introducing the recombinant vector into a host, PA1 C. cultivating the host transformed with the recombinant vector to produce the polypeptide, PA1 D. collecting the cultured cells and extracting the polypeptide produced from them#and PA1 E. purifying the polypeptide by conventional purifying methods for proteins.
The processes disclosed in these documents are characterized by involving purification of TNF from body fluids (e.g., blood) or tissues of rabbits, mice, hamsters or guinea pigs as raw materials. The products, however, are not so clearly defined, and it is evident that various restrictions are imposed on these processes in regard to the supply of raw materials and the purities of the final products.
In clinical application as a potent antitumor agent, TNF originated from humans is desirable in consideration of immunogenicity. The above-described processes, however, cannot be applied to the production of human TNF.
The following have so far been reported on an antitumor cytotoxin originated from humans: cytotoxins produced from human peripheral monocutes and human myelocytic monocytic leukemia cells [Matthews, Immunology, 44, 135 (1981)], from the adherent cells in human peripheral blood cells [Reed, et al., J. Immunol., 115, 395 (1975)], and from human B cell lines [Williamson, et al., Proc. Nat. Acad. Sci., U.S.A., 80, 5397 (1983)). These substances have cytotoxic activity, but are not defined clearly.